Research Report
Cecilia McIntosh
Department of Biological Sciences
East Tennessee State University
Since my undergraduate days, I have been fascinated with regulation and
metabolism, and I am continually amazed by the "bio-logic" of this
regulation - once it is elucidated! The research experiences leading to
my Ph.D., followed by a postdoctoral appointment, have had a profound
impact on the focus of two current research projects in my laboratory.
The theme of my doctoral work with Dick Mansell at the University of
South Florida, as well as earlier work in his laboratory, was
understanding the dynamics of patterns of accumulation of the citrus
bitter principles limonin (a triterpenoid) and naringin (a flavonoid). My
doctoral research came from an interest in elucidating the biosynthesis
of naringin from naringenin, and involved isolation and characterization
of a flavanone-specific 7-O-glucosyltransferase from grapefruit
seedlings. Naringin can comprise up to 75% of the dry weight of young
leaves and fruit, and synthesis/accumulation appears to be under
developmental and/or tissue-specific controls. Information on the
biochemical and molecular regulation of flavonoid biosynthesis has been
rapidly evolving in the literature, and a coordinated picture is
developing. Currently in my laboratory we are in the initial stages of
addressing one potential piece of the puzzle: the flavanone "branch"
point from the aspect of flavanone glycosylation vs. flavanone conversion
into other flavonoids (supported by a USDA Seed Grant).
At Iowa State University's botany department with David Oliver, I spent
three years studying the biochemistry and molecular biology of plant
mitochondrial proteins. Characterizing the phosphate and tricarboxylate
metabolite transporters in pea mitochondria gave me my first experience in
working with membrane proteins. The technical difficulty in achieving
even a rudimentary level of purification of integral membrane proteins is
significant! One intriguing aspect of the mitochondrial research was
comparing characteristics of the plant proteins with their more completely
studied animal counterparts. For example, phosphate transporters are
quite active in both plants and animals, but the tricarboxylate
transporter has a lower level of activity in plants. Structure
comparisons from amino acid sequence information, inferred from putative
coding regions in genomic clones, revealed some differences, although I
did not obtain a complete clone from either cDNA or genomic libraries in
the time available.
I also isolated, purified and characterized NAD-dependent citrate
dehydrogenase (NAD-ICDH) from the mitochondrial matrix of peas. NAD-ICDH
is a TCA cycle regulatory enzyme. Once again, the characteristics of the
plant enzyme showed some interesting differences as compared to those
from non-plant sources. For example, bovine NAD-ICDH is allosterically
regulated by ADP/ATP levels and is most active when ADP levels are high.
Pea NAD-ICDH is not affected by ADP/ATP but is allosterically inhibited
by NADPH. I find it intriguing that while the same enzyme is regulatory
in both organisms, the biochemical mode of regulation is different and
reflects the physiology of the particular organism. Recently I have been
working on the characterization of a membrane associated pool of NAD/ICDH
in pea mitochondria. This pool is strongly associated with the membrane
and may represent another form of the enzyme.
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